In a healthy human body, there is a balance between the delivery and removal of cholesterol. When people have a high level of low-density lipoprotein (LDL) and low level of high-density lipoprotein (HDL), the imbalance results in more cholesterol being deposited in the arteries than that being removed (van Dam, M. J. et al. 2002, Lancet 359: 37-42)). Atherosclerosis, the narrowing or blocking of arteries, is a consequence of the repeated deposit of cholesterol, termed plaque (Major, A. S. et al. 2001, Arterioscler. Thromb. Vasc. Biol. 21: 1790-1795)).
Lipoproteins can be separated into atherogenetic and vasoprotective lipoproteins. Atherogenetic lipoproteins are generally all apolipoprotein (Apo) B-containing lipoprotein such as very-low-density lipoprotein (VLDL), intermediate (IDL), low (LDL) or lipoprotein (Lp(a)), whereas vasoprotective lipoproteins are Apo AI containing, such as HDL.
Apo AI, the major protein constituent of HDL, plays a critical role in cholesterol homeostasis. Clinical and population-based studies have demonstrated a remarkable inverse correlation between cardiovascular disease and plasma HDL levels, suggesting Apo AI and HDL help to serve a protective role against atherogenesis (Rubins, H. B. et al. 1993, Am. J. Cartiol. 71: 45-52)). Studies of transgenic mice (Rubin, E. M. et al. 1991, Nature 353: 265-267)) and rabbits (Duverger, N. et al. 1996, Circulation 94: 713-717)) susceptible to atherosclerosis have shown that expression of human Apo AI inhibits the development of atherosclerosis. This effect may be related to its efficient promotion of cholesterol efflux from cells (Castro, G. et al. 1997, Biochemistry 36: 2243-2249)), the first step in the process of ‘reverse cholesterol transport’ (RCT) (Glomset, J. A. 1968, J Lipid Res. 9: 155-167)). Apo AI modulates this process by being a preferential acceptor of cellular cholesterol (Rothblat, G. H. et al. 1999, J Lipid Res. 40: 781-796)), increasing the activity of lecithin-cholesterol-acyl-transferase (LCAT) esterification of HDL-associated cholesterol several-fold (Jonas, A. 1991, Biochim. Biophys. Acta 1084: 205-220; Mahley, R. W. et al. 1984, J Lipid Res. 25: 1277-1294)), and transporting LCAT-derived cholesteryl esters to the liver (Morrison, J. R. et al. 1992, J. Biol. Chem. 267: 13205-13209)).
Unlike synthetic antihyperlipidemics, such as LIPITOR® (atorvastatin calcium) that act to lower lipid levels in the body by inhibiting the synthesis of cholesterol (Alaupovic, P. et al. 1997, Atherosclerosis 133: 123-133)), an infusion of purified Apo AI stimulates cholesterol efflux from tissues into plasma (Navab, M. et al. 2002, Circulation 105: 290-292)). This suggests that Apo AI could stimulate cholesterol efflux from foam cells in the arterial wall and induce regression of atherosclerotic plaque, effectively ‘cleaning out’ the arteries.
In humans, Apo AI is synthesized in liver and intestinal cells as a non-glycosylated pre-pro-protein (Gordon, J. I. et al. 1983, J. Biol. Chem. 258: 4037-4044)). The 18 amino acid pre-segment is removed before the protein leaves the cell and the 6 amino acid pro-segment is cleaved post secretion by an unknown protease in the plasma, leaving the mature 243 amino acid protein (Saku, K. et al. 1999, Eur. J. Clin. Invest. 29: 196-203)).
The Apolipoprotein A-IMilano (Apo AI-M) is the first described molecular variant of human Apo AI (Franceschini, G. et al. 1980, J. Clin. Invest. 66: 892-900)). It is characterized by the substitution of Arg 173 with Cys (Weisgraber, K. H. et al. 1983, J. Biol. Chem. 258: 2508-2513)). The mutant apolipoprotein is transmitted as an autosomal dominant trait and 8 generations of carriers have been identified (Gerli, G. C. et al. 1984, Hum. Hered. 34: 133-140)).
The status of the Apo AI-M carrier individual is characterized by a remarkable reduction in HDL-cholesterol level. In spite of this, the affected subjects do not apparently show any increased risk of arterial disease; indeed, by examination of the genealogic tree it appears that these subjects may be “protected” from atherosclerosis.
The mechanism of the possible protective effect of Apo AI-M in the carriers seems to be linked to a modification in the structure of the mutant apolipoprotein, with the loss of one alpha-helix and an increased exposure of hydrophobic residues (Franceschini, G. et al. 1985, J. Biol. Chem. 260: 16321-16325). The loss of the tight structure of the multiple alpha-helices leads to an increased flexibility of the molecule, which associates more readily with lipids, compared to normal A-I. Moreover, apolipoprotein/lipid complexes are more susceptible to denaturation, thus suggesting that lipid delivery is also improved in the case of the mutant.
The therapeutic use of Apo AI and the Apo AI-M mutant is presently limited by the lack of a method allowing the preparation of said apolipoproteins in sufficient amount and in a suitable form. In particular, the recombinant production of Apo AI has been shown to be very difficult due to its amphiphilic character, autoaggregation, and degradation (Schmidt, H. H. et al. 1997, Protein Expr. Purif. 10: 226-236). At the time of the present invention, recombinant human Apo AI has been expressed in vitro in two eukaryotic systems: Baculovirus transfected Spodoptera frugiperda (Sf9) cells (Sorci-Thomas, M. G. et al. 1996, J. Lipid Res. 37: 673-683) and stably transfected Chinese hamster ovary (CHO) cells (Forte, T. M. et al. 1990, Biochim. Biophys. Acta 1047: 11-18; Mallory, J. B. et al. 1987, J. Biol. Chem. 262: 4241-4247). In the baculovirus system, once the cells are successfully transfected, there is an in-depth screening process before cells with the correct construct can be used for expression. Similarly, CHO cell colonies must undergo a screening process to find stably transfected, high expressing colonies. Additionally, both of these cell types require a relatively long period of time before significant expression is achieved and a much higher level of maintenance than bacteria.
Recombinant expression of proteins in bacterial systems is generally attractive because it can produce large amounts of pure protein quickly and economically There are several reports of Apo AI expression in transformed Escherichia coli (E. coli); however, while certain recent improvements in expression levels have been made (Ryan, R. O. et al. 2003, Protein Expr. Purif. 27: 98-103) in general, these methods provide relatively low yields or the undesirable presence of extraneous affinity tags or secretion signals (Bergeron, J. et al. 1997, Biochim. Biophys. Acta 1344: 139-152; L1, H. H. et al. 2001, J. Lipid Res. 42: 2084-2091; McGuire, K. A. et al. 1996, J. Lipid Res. 37: 1519-1528). Moreover, E. coli endotoxins are known to form particularly strong complexes with apolipoproteins (Emancipator et al. (1992) Infect. Immun. 60: 596-601). Reduction or elimination of the toxicity associated with these E. coli endotoxins in pharmaceutical preparations of apoliproteins is highly desirable. The removal of these endotoxins, while technically feasible, involves complex and expensive protein purification methodologies (U.S. Pat. No. 6,506,879) without fully eliminating the human health risk.
The use of plants as bioreactors for the large scale production of recombinant proteins is known to the art, and numerous proteins, including human therapeutic proteins, have been produced. For example, U.S. Pat. Nos. 4,956,282, 5,550,038 and 5,629,175 disclose the production of γ-interferon in plants; U.S. Pat. Nos. 5,650,307, 5,716,802 and 5,763,748 detail the production of human serum albumin in plants and U.S. Pat. Nos. 5,202,422, 5,639,947 and 5,959,177 relate to the production of antibodies in plants. One of the significant advantages offered by plant-based recombinant protein production systems is that by increasing the acreage of plants grown, protein production can be inexpensively scaled up to provide for large quantities of protein. By contrast, fermentation and cell culture systems have large space, equipment and energy requirements, rendering scale-up of production costly. However, despite the fact that the use of plants as bioreactors is amply documented, and despite the above mentioned therapeutic applications of apolipoproteins, the prior art provides no methods for the production of apolipoproteins in plants.
In order to offer a practical alternative to the fermentation and cell culture based systems, it is important that plants remain healthy and that apolipoproteins accumulate to significant levels in the plants. In view of the inherent property of apolipoproteins to associate with lipids, recombinantly expressed apolipoproteins may associate with the endogenous plant lipids and thereby interfere with the plant's lipid metabolism. Thus recombinant expression of apolipoproteins may affect the health of the plant. Alternatively, the recombinantly expressed apolipoprotein may fail to accumulate to effective levels, as protective mechanisms may result in degradration of the apolipoprotein. It thus is unclear whether and how the synthetic capacity of plants may be harnessed to achieve the commercial production of apolipoproteins in plants.
Thus in view of the shortcomings associated with the methods for the recombinant production of apolipoproteins by the prior art, there is a need in the art to improve methods for the production of apolipoproteins.